Escherichia coli (E. coli) is a gram negative bacterium which occurs naturally in the intestinal tract of warm-blooded animals. The presence of this bacterium in water is a reliable indicator of fecal contamination. If E. coli bacterium is present in a given sample of water, it is also possible that Salmonella, Shigella, Vibrio, enteric viruses, and intestinal parasites are also present in the sample.
No reliable single step process is known to detect the presence of E. coli in a sample of water containing mixed bacteria. Currently a two-step process is used to detect this microorganism. In this process a broth culture is inoculated with the water sample and then incubated at 35.degree. C and examined for gas production after 24 and 48 hours. The gas is detected in Durham tubes, which are small test tubes inverted in the culture broth and filled with the culture medium. As the gas is being produced, it is collected in the Durham tubes. The gas becomes visible as a bubble in the Durham tube. It is presumed that the tubes which contain gas bubbles have detected E. coli. Growth from these tubes are then transferred to fresh broth in which are immersed other Durham tubes. The specimens are incubated at 44.5.degree. C for an additional 24 hours and those tubes which produce gas at 44.5.degree. C are termed "Positive". Under present practice, it is presumed the gas producing tubes represent confirmation that E. coli was present in the water sample tested. Over the past 20 years it has become evident to those working in the art that many of the Durham tubes which indicated "Positive" results did not actually contain E. coli. The tubes were rendered "Positive" by microorganisms such as Klebsiella, Enterobacter and Citrobacter. Because these microorganisms are also found in the intestinal tract, the term "Fecal Coliform" was coined to describe microorganisms which produce gas at 44.5.degree. C in the aforementioned two-step process. However, it is now known that several of the Klebsiella and Enterobacter organisms which give "Positive" results in the current tests at 44.5.degree. C are not of fecal origin, but are normal inhabitants of soil, water, and plants.
We have discovered a process which does not depend on gas production to detect E. coli in water samples. Our process involves neither transfer of positive Durham tube cultures to a second culture broth nor a second incubation step. We have discovered a culture medium and a single step method wherein the medium is inoculated with the water sample, incubated at about 35.degree. to about 38.degree. C for about 4 to about 6 hours, and then incubated at about 44.degree. to about 46.degree. C for about 10-12 hours. Positive results are indicated by means of a change in color of a pH indicator solution incorporated into the medium. The entire test can be completed within 16 to 20 hours, whereas current methods of detection require from 36 to 48 hours. Our test requires only one tube per culture whereas current methods require two tubes per culture. Our test also eliminates the use of Durham tubes, which are both costly and time consuming. Furthermore our test gives a far better percentage indication of E. coli than do current methods because most Klebsiella and Enterobacter species which normally grow and produce acid (gas) at 44.5.degree. C are inhibited by para-coumaric acid and acetazolamide, and thus do not grow at this elevated temperature.